The Role Of TLR3 Of Spinal Astrocytes In Neuropathic Pain

Part 1 TLR3 expression in spinal astrocytes in rats following spinal nerveligationObjective To investigate the role of changes in TLR3 expression in spinalastrocytes induced by spinal nerve ligation(SNL).Methods Sixty-six male SD rats weighing 180～250 g were randomly divided into 2groups: SHAM group(n=30) and SNL group(n=36). The animals were anesthetizedwith intraperitoneal 10% Chloral Hydrate 0.3～0.35ml/100g. Neuropathic pain wasinduced by L₅ spinal nerve ligation. The right spinal nerve was exposed and ligatedwith 4-0 silk thread. In sham operation group the right spinal nerve was exposed butnot ligated. Pain threshold was estimated by measuring paw withdrawal pressurethreshold and latency with Von Fery filament stimulation at 1 d before operation(baseline) and 1, 3, 5, 7, 10, 14, 21, 28 day after SNL. The animals were terminatedon the 7th day after SNL and the lumbar segment of the spinal cord was removed and the expression of TLR3 and GFAP positive cells in the spinal dorsal horn weredetermined by immunofluorescence double-staining.The rats were terminated at 3, 7,14, 21, 28d after operation in each group and the L_4～5 segment of the spinal cord wasremoved for determination of expression of TLR3 mRNA by RT-PCR.Result In SNL group PWPT and PWL was significantly decreased compared withthose in SHAM group (P＜0.05). Immunofluoresence double-staining indicated thatTLR3 and GFAP were colocalizated in the spinal astrocytes in SNL group. Theexpression of TLR3 mRNA in the ipsilateral spinal dorsal horn was significantlyincreased in SNL group compared with SHAM group (P＜0.05).Conclusion Up-regulation of TLR3 gene expression in the spinal dorsal horn maybe involved in the mechanism of SNL-induced neuropathic pain.Part 2 The role of TLR3 of spinal astrocytes in neuropathic painStudy 1 Neuropathic pain induced by intrathecal injection of poly(I:C)Objective To investigate the role of TLR3 of spinal astrocytes in neuropathic paininduced by intrathecal(i.t.) poly(I:C).Methods: 126 male SD rats weighing 180～250 g were randomly divided into 3groups: group C (n=42),control group; group NS(n=42), NS (0.5ml/kg,i.t.), once aday for 7 consecutive days; group NP(n=42), minocycline(40mg/kg,i.p.)+ Poly(I:C)(0.5mg/kg, i.t.), once a day for 7 consecutive days. Pain threshold was estimated bymeasuring paw withdrawal pressure threshold and latency with Von Fery filamentstimulation at -1 (1 d before operation), 1, 3, 5, 7, 10, 14, 21, 28 day after injection.The animals were killed on the 7th day after injection and the lumbar segment of thespinal cord was removed and the expression of GFAP positive cells in the spinaldorsal horn were determined by immunohistochemistry ABC method. The rats were killed at -1, 1, 7, 14, 21, 28d after injection and the L_4-5 segment of the spinal cordwas removed for determination of expression of IL-6, TNF-a, IFN-β, IP-10 and TLR3mRNA by RT-PCR. To test whether the upregulation of the IL-6 and IP-10 in protein,we measured IL-6 and IP-10 protein levels in the media by ELISA.Result In group NP, PWPT was significantly decreased compared with group C andNS (P＜0.05). Immunohistochemistry indicated that staining of GFAP immunoreactivecells in group NP much more than group C and NS(P＜0.05).The expressionof TLR3 mRNA in the spinal dorsal hom was significantly increased in group NPcompared with group C and NS (P＜0.05). Significantly higher spinal expression ofmRNA for IL-6, TNF-a, IFN-βand IP-10 was observed at day 7 after injection ofpoly(I:C) compared with saline (P＜0.05). IL-6 (9.5ng/g) and IP-10 (12.3ng/g) proteinlevels in poly(I:C)-stimulated rats were significantly elevated at day 7 (P＜0.05) afterintrathecal injection compared with saline control animals(P＜0.05).Conclusion TLR3 of spinal astrocytes may be involved in the mechanism ofpoly(I:C)-induced neuropathic pain.Study 2 Analgesic effect of intrathecal antisence oligodeoxynucleotide of TLR3on rats following spinal nerve ligationObjective To investigate the analgesic effect of TLR3 expression of spinalastrocytes on neuropathic pain induced by spinal nerve ligation(SNL).Methods Male SD rats weighing 180～250 g were included in this experiment.Catheter was placed intrathecally with a tip located at lumbo-sacral segment of spinalcord for intrathecal(i.t.) drug administration in all animals. Neuropathic pain wasinduced by ligation of right L₅ spinal nerve with 4-0 silk thread. In Partâ… , 108 animalswere randomly and equally divided into 3 groups: group A (n=36), normal saline(NS)(20μl,i.t.)+SNL; group B (n=36), mismatch oligodeoxynucleotide (MM-ODN) (20μg/d,i.t.) +SNL; group C (n=36), antisense oligodeoxy- nucleotide (AS-ODN)(20μg/d,i.t.)+SNL, once a day for 7 consecutive days. In Partâ…¡,on the 7^th day afterSNL another 108 rats were randomly and equally divided into 3 groups: group D(n=36), SNL + NS(20μl,i.t.); group E (n=36), SNL + MM-ODN(20μg/d,i.t.); group F(n=36), SNL +AS-ODN (20μg/d,i.t.), once a day for 7 consecutive days. Painthreshold was estimated by measuring paw withdrawal pressure threshold(PWPT) andlatency(PWL) with Von Fery filament stimulation at -1 (1 d before operation), 1, 3, 5,7, 10, 14 day in Partâ… and -1, 7, 10, 12, 14 day in Partâ…¡after SNL, respectively. Theanimals were killed on the 7^th(Partâ… ) or 14^th(Partâ…¡) day after SNL and lumbarsegments of spinal cord were removed and immunohisto-chemistry ABC method wasused to determine the expression of GFAP positive cells in the spinal dorsal horn. Therats were killed at -1, 3, 7, 14 day in Partâ… and -1,7, 10, 14 day in Partâ…¡afteroperation in each group and the L_4-5 segment of the spinal cord was removed fordetermination of expression of TLR3 and IL-6 mRNA by RT-PCR. To test thevariance of the IL-6 and IP-10 in protein, we measured IL-6 and IP-10 protein levelsin the media by ELISA.Results Partâ… : In group C, PWPT was significantly increased compared with group A(P＜0.05), PWL has no significant difference at different time (P＞0.05). Immunohisto-chemistryindicated that the relative area of staining of GFAP immuno-reactive cellsin group C decreased 46.4% and 45.7% (P＜0.05) respectively, when compared withthat in group A and B. The expression of TLR3 and IL-6 mRNA, IL-6 and IP-10protein was significantly inhibited in group C (P＜0.05). Partâ…¡: There was nosignificant difference in the above-mentioned markers among group D, group E andgroup F (P＞0.05).Conclusion TLR3 of spinal astrocytes in the spinal dorsal horn may be involved inthe development of SNL-induced neuropathic pain. Part 3 Activation of JNK in spinal cord dorsal horn following neuropathic painmediated by TLR3Objective To investigate whether the intracellular signal transduction pathway isinvolved in the mechanisms of neuropathic pain, we examine the activation of c-JunN-terminal kinase (JNK) in the dorsal horn of the spinal cord in a rat model afterintrathecal(i.t.) poly(I:C) or antisense oligodeoxynucleotide of TLR3.Methods Male SD rats weighing 180～250 g were used in this experiment. In allanimals catheter was placed intrathecally with the tip located at lumbo-sacral segmentof the spinal cord for intrathecal drug administration. Neuropathic pain was inducedby right L₅ spinal nerve ligation with 4-0 silk thread. 24 animals were randomlydivided into 4 groups (n=6 each): group A, Poly(I:C) (0.5ml/kg,i.t.); group B,SP600125 50nmol + Poly(I:C) (0.5ml/kg,i.t.); group C, mismatch oligodeoxynucleotide(MM-ODN) (20μg/d,i.t.) +SNL; group D, antisense oligodeoxynucleotide (ASODN)(20μg/d,i.t.) +SNL, once a day for 7 consecutive days. Pain threshold wasestimated by measuring paw withdrawal pressure threshold(PWPT) and latency(PWL)with Von Fery filament stimulation at -1(1 d before operation), 1, 3, 5, 7 day in groupA and B after i.t. or in group C and D after SNL. The animals were killed on the7^thday in group A and B after i.t. or in group C and D after SNL and the lumbarsegment of the spinal cord was removed and the expression of pJNK positive cells inthe spinal dorsal horn were determined by immuno fluorescence histochemistry.Results Poly(I:C) induced dramatic mechanical allodynia from day 1 to day 7 afteri.t., An increase in the phosphorylation of JNK in the dorsal horn of the spinal cordwas also observed after i.t.; Intrathecal infusion of a JNK peptide inhibitor, SP600125,did not affect normal pain responses but potently prevented poly(I:C)-inducedmechanical allodynia and the activation of JNK. Intrathecal infusion of antisense oligodeoxynucl-eotide of TLR3 also potently prevented SNL-induced mechanicalallodynia and the activation of JNK.Conclusion Activation of JNK in spinal cord dorsal horn may be involved in themechanism of TLR3-mediated neuropathic pain in rats.